The association of human adipose angiotensinogen gene expression with abdominal fat distribution in obesity.

OBJECTIVE: To investigate in obese subjects the relationship between angiotensinogen gene expression in the abdominal omental and subcutaneous adipose tissue on the one hand and body fat distribution as measured by waist-to-hip ratio (WHR) on the other hand and to compare angiotensinogen gene expression between the two adipose tissue regions. SUBJECTS: Twenty obese subjects undergoing weight reduction surgery with adjustable gastric banding (12 men, eight women; WHR 0.89-1.09; body mass index (BMI) 29-51 kg/m2, age 26-54 y). MEASUREMENTS: Omental and subcutaneous adipose angiotensinogen mRNA and 18S ribosomal RNA (reference gene) levels were measured by competitive quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Angiotensinogen mRNA levels were one-third higher in the omental than in the subcutaneous adipose tissue region (P=0.02). The 18S rRNA levels did not differ significantly between the two adipose tissue regions. WHR correlated positively and significantly with angiotensinogen mRNA in both the subcutaneous and the omental adipose tissue (r=0.5). This relationship was independent of age and BMI. However, WHR did not correlate with 18S rRNA in any of the adipose tissue regions. CONCLUSION: The angiotensinogen gene in adipose tissue might be involved in the development of upper-body obesity.

Definition of a subset of human peripheral blood mononuclear cells that are permissive to human cytomegalovirus infection.

The identity of cells responsible for transmission of human cytomegalovirus (HCMV) in blood products or bone marrow transplants is unknown. We have tested the capacity of HCMV to in vitro infect human peripheral blood mononuclear cells (PBMC) from healthy donors and found that certain PBMC are permissive to HCMV infection. In vitro-infected viable cells were double stained for surface expression of different HMCV proteins and for cell-type-specific antigens to allow the identification of sensitive cells. All analysis were performed on viable cells, using HCMV-specific monoclonal antibodies and automated flow cytofluorimetry. PBMC were infected either with the laboratory-adapted HCMV strain AD169 or with a virus isolate obtained from a viremic patient. Up to 25% of all PBMC could express the major immediate-early antigen as well as the pp65 antigen, known at the lower matrix protein. Infected cells were mainly CD14+ monocytes, but also a small population of large CD8+ cells were susceptible to HCMV infection. CD19+ B lymphocytes were resistant to HCMV infection. Different populations of infected cells were enriched by using Dynabeads coated with cell-type-specific antibodies, and the presence of infectious virus was demonstrated by incubating the selected and sonicated cell material on human fibroblasts. Only material from infected monocytes and from CD3+ CD8+ cells gave rise to HCMV-specific plaques. The presence of HCMV mRNA as a sign of active viral transcription of the major immediate-early and late pp150 genes in infected cells was demonstrated by using nested reversed polymerase chain reaction. A common denominator was found for all cells that could be infected with HCMV. The CD13 antigen, a 130- to 150-kDa integral membrane protein identical to the enzyme aminopeptidase N, was expressed on all HCMV-permissive cells.

Identification of blood mononuclear cells permissive of cytomegalovirus infection in vitro.

We conclude that both the experimental strain Ad169 and a CMV isolate obtained from a patient could infect PBMCs in vitro. The identity of infected cells was established as CD14+ monocytes and a small population of CD3/CD8+ large granular lymphocytes. No evidence of sensitivity to infection was obtained in small lymphocytes, in either the B or T cell population. Furthermore, flow cytometric analysis of cells expressing CMV-encoded antigens is a sensitive assay, as is the demonstration of viral RNA using reversed PCR and nested primer pairs. Since three different types of analyses all indicated active production of structural CMV antigens in infected cells, we conclude that monocytes and some CD8+ large lymphocytes might serve as reservoirs for latent CMV infection and might be responsible for the transfer of CMV infection. Presently, it cannot be determined whether PBMCs are indeed sensitive to a primary infection. Direct sequencing of virus isolates from in vitro-infected cells will have to be carried out to settle this issue. However, we favor the explanation that CMV causes a primary in vitro infection, since, as discussed above, various activating substances can induce the expression of CMV-encoded antigens in blood cells from seropositive donors. Finally, we found that the presence of the CD13 marker was a common denominator of all cells sensitive to CMV infection. We are presently attempting to elucidate whether the CD13 molecule is instrumental in the infection of cells by CMV.

Interleukin 4 instructs uncommitted B lymphocytes to switch to IgG1 and IgE.

Mouse interleukin 4 (IL 4) is a T cell-produced lymphokine with multiple effects on different cells types of the hematopoietic lineages. IL 4 has pronounced effects on B lymphocytes, where it induces high levels of IgG1 and IgE secretion in lipopolysaccharide-stimulated cultures that would otherwise secrete predominantly IgG3 and IgG2b (of the non-IgM isotypes). An important question is how IL 4 exerts its effect. Two main possibilities exist: (a) IL 4 instructs uncommitted B lymphocytes to IgG1 and IgE production; (b) IL 4 selects and expands an already precommitted B cell. In this study we show, by the use of limiting dilution analysis, that IL 4 dramatically increases the precursor frequency of IgG1 and IgE-secreting cells with no significant effect on the clone size, clearly suggesting that IL 4 instructs uncommitted B cells to switch to IgG1 and IgE. The fraction of total Ig precursors that can switch to the two isotypes is furthermore high. The high precursor frequency for IgE obtained in the presence of IL 4 further demonstrates that IL 4 is an important modulator of IgE responses.

IgG1 induction factor: a single molecular entity with multiple biological functions.

A cDNA clone coding for the murine IgG1 induction factor has been isolated. The translation products directed by this clone were analyzed in different biological assays. The data obtained show that the IgG1 induction factor: Is involved in the regulation of IgG responses, by increasing IgG1 and decreasing IgG3 and IgG2b secretion; Induces hyper-Ia expression on resting B lymphocytes; Synergizes with anti-Ig in inducing DNA synthesis in resting B lymphocytes; Synergizes with DxS in inducing DNA synthesis by B lymphocytes; It induces DNA synthesis by either the T cell line CTL-L or Con-A blasts. Thus, this lymphokine in addition to IgG1 inducing activity has also BSF-1, BCGF-II and TCGF like activities. The fact that a single molecule can perform all the above listed functions has implications for our view of lymphocyte activation. It indicates that considering the B cell response as an ordered series of independently controlled events, is an oversimplified view of the dynamic process through which B cells are activated and also indicate the functional interconnection of the different elements of the immune system.

IgG1 induction factor.

The IgG1 induction factor elevates the IgG1 response and reduces the IgG2b and IgG3 response in LPS-stimulated spleen cells. The factor is a lymphokine produced by T cells. The precursor frequency for cells secreting the IgG1 induction factor is at least tenfold lower as compared to those secreting interleukin 2. Some biochemical properties of the lymphokine are listed. The effects of gamma interferon in B cell-stimulated cultures are shown. Isolation of a cDNA clone coding for the IgG1 induction factor has been achieved and results of these studies are reviewed. Evidence is given that this lymphokine is the same as B cell stimulating factor 1, and we propose that it be renamed interleukin 4. Finally, the possible mechanisms of interleukin 4 are discussed.

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II.

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.

Secretion of IgG1 induction factor by T cell clones and hybridomas.

IgG1 induction factor elevates the IgG1 response induced by lipopolysaccharide and suppresses the lipopolysaccharide-induced IgG3 and IgG2b responses in cultures of mouse spleen cells. We have developed new T cell lines secreting this factor by cloning mixed lymphocyte culture populations. Using supernatants of one of these T cell lines it was found that the assay is quantitative, reproducible and accurate, both when induction of IgG1 as well as reduction of IgG3 and IgG2b were measured. Using this analysis, different conditions to induce maximal production of the factor were tested. The cell line was thereafter used as fusion partner with a T cell lymphoma. The hybrids were selected in the presence of T cell growth factor and all of them secreted IgG1 induction factor.

Partial biochemical characterization of IgG1-inducing factor.

IgG1 induction factor elevates the IgG1 and suppresses the IgG3 and IgG2b responses in lipopolysaccharide-stimulated murine spleen cell cultures. By the use of a quantitative assay, it was found that the three activities, induction of IgG1 and reduction of IgG3 and IgG2b synthesis, were found in the same fractions after different chromatographic procedures, suggesting that the same molecule was responsible for the effects. The factor was precipitated by 60-90% saturation of ammonium sulfate and was sensitive to proteolytic cleavage and to treatment with a buffer of pH 10. It had an apparent molecular mass of 20 kDa as judged by gel filtration chromatography and was separated into two peaks after isoelectric focusing, pI 7.4-7.2 and 6.4-6.2, respectively. Finally it was weakly hydrophobic and negatively charged at pH 7.55. These characteristics indicate that the factor is different from many previously characterized lymphokines and similar or identical to the B cell stimulating factor-1 (BSF-p1). The relevance of these findings to the mechanism of the immunoglobulin class switch is discussed.

Limited life span of extensively proliferating B cells: no evidence for a continuous class or subclass switch.

The polyclonal B cell activators (PBA) dextran sulphate (DxS) and lipopolysaccharide (LPS) induce a marked synergistic response in mouse splenic B cells. Such activated B cell blasts could be maintained in culture for a total period of 15 to 20 days, provided the cells were subcultured repeatedly in fresh medium containing the PBA. After this time period activation could no longer be achieved. The B cell blasts responded at all times better to LPS plus DxS than to either PBA added alone. Furthermore, LPS-induced B cell blasts also responded better to LPS plus DxS than to LPS alone. Activated cells secreted IgM and switched to IgG3 and IgG2b secretion. At the end of the culture period, approximately one cell out of four secreted IgG. The kinetics of IgG3 and IgG2b production were similar, and the fraction of cells secreting these subclasses increased at similar rates throughout the culture period. We conclude that high-rate secreting and proliferating B cells have a limited life span. Furthermore, most activated B cells do not seem to go through a continuous subclass switch but keep on secreting the subclass they originally switched to.

A synergistic polyclonal response to dextran sulphate and lipopolysaccharide: immunoglobulin secretion and cell requirements.

The effects of the simultaneous addition of lipopolysaccharide (LPS) and dextran sulphate (DxS) were studied in a low-cell density culture system, which enables extensive cell proliferation and high immunoglobulin secretion. Using these two mitogens, a synergistic response was observed, with regard to both cell division and IgM secretion. However, only a very low IgG production could be detected. This was caused by the extensive cell proliferation, leading to suboptimal culture conditions. Thus, when the blasts were recultured at a lower density or when a lower initial cell concentration was used, a high IgG response was obtained. The synergistic response induced by LPS plus DxS was independent of T cells. Furthermore, no apparent need for phagocytic cells was found. Both the LPS- and the LPS plus DxS-induced activation led to a switch, preferentially to IgG2b and IgG3 secretion. This subclass pattern was not changed when the cultures were lacking functional T cells.